Dataset Information

The bias associated with amplicon sequencing does not affect the quantitative assessment of bacterial community dynamics.

ABSTRACT: The performance of two sets of primers targeting variable regions of the 16S rRNA gene V1-V3 and V4 was compared in their ability to describe changes of bacterial diversity and temporal turnover in full-scale activated sludge. Duplicate sets of high-throughput amplicon sequencing data of the two 16S rRNA regions shared a collection of core taxa that were observed across a series of twelve monthly samples, although the relative abundance of each taxon was substantially different between regions. A case in point was the changes in the relative abundance of filamentous bacteria Thiothrix, which caused a large effect on diversity indices, but only in the V1-V3 data set. Yet the relative abundance of Thiothrix in the amplicon sequencing data from both regions correlated with the estimation of its abundance determined using fluorescence in situ hybridization. In nonmetric multidimensional analysis samples were distributed along the first ordination axis according to the sequenced region rather than according to sample identities. The dynamics of microbial communities indicated that V1-V3 and the V4 regions of the 16S rRNA gene yielded comparable patterns of: 1) the changes occurring within the communities along fixed time intervals, 2) the slow turnover of activated sludge communities and 3) the rate of species replacement calculated from the taxa-time relationships. The temperature was the only operational variable that showed significant correlation with the composition of bacterial communities over time for the sets of data obtained with both pairs of primers. In conclusion, we show that despite the bias introduced by amplicon sequencing, the variable regions V1-V3 and V4 can be confidently used for the quantitative assessment of bacterial community dynamics, and provide a proper qualitative account of general taxa in the community, especially when the data are obtained over a convenient time window rather than at a single time point.

SUBMITTER: Ibarbalz FM

PROVIDER: S-EPMC4055690 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The bias associated with amplicon sequencing does not affect the quantitative assessment of bacterial community dynamics.

Ibarbalz Federico M FM Pérez María Victoria MV Figuerola Eva L M EL Erijman Leonardo L

PloS one 20140612 6

The performance of two sets of primers targeting variable regions of the 16S rRNA gene V1-V3 and V4 was compared in their ability to describe changes of bacterial diversity and temporal turnover in full-scale activated sludge. Duplicate sets of high-throughput amplicon sequencing data of the two 16S rRNA regions shared a collection of core taxa that were observed across a series of twelve monthly samples, although the relative abundance of each taxon was substantially different between regions. ...[more]

PMID: 24923665

Similar Datasets

Project description:Fermented fruits and vegetables play an important role in safeguarding food security world-wide. Recently, robust sequencing-based microbial community analysis platforms have improved microbial safety assessment. This study aimed to examine the composition of bacteria and evaluate the bacterial safety of fermented fruit products using high-throughput 16S-rRNA metagenomic analysis. The operational taxonomic unit-based taxonomic classification of DNA sequences revealed 53 bacterial genera. However, the amplicon sequencing variant (ASV)-based clustering revealed 43 classifiable bacterial genera. Taxonomic classifications revealed that the abundance of Sphingomonas, which was the predominant genus in the majority of tested samples, was more than 85-90% among the total identified bacterial community in most samples. Among these identified genera, 13 low abundance genera were potential opportunistic pathogens, including Acinetobacter, Bacillus, Staphylococcus, Clostridium, Klebsiella, Mycobacterium, Ochrobactrum, Chryseobacterium, Stenotrophomonas, and Streptococcus. Of these 13 genera, 13 major opportunistic pathogenic species were validated using polymerase chain reaction. The pathogens were not detected in the samples of different stages and the final products of fermentation, except in one sample from the first stage of fermentation in which S. aureus was detected. This finding was consistent with that of ASV-based taxonomic classification according to which S. aureus was detected only in the sample from the first stage of fermentation. However, S. aureus was not significantly correlated with the human disease pathways. These results indicated that fermentation is a reliable and safe process as pathogenic bacteria were not detected in the fermentation products. The hybrid method reported in this study can be used simultaneously to evaluate the bacterial diversity, their functional predictions and safety assessment of novel fermentation products. Additionally, this hybrid method does not involve the random detection of pathogens, which can markedly decrease the time of detection and food safety verification. Furthermore, this hybrid method can be used for the quality control of products and the identification of external contamination.

Project description:Bacterial endosymbionts that produce important phenotypic effects on their hosts are common among plant sap-sucking insects. Aphids have become a model system of insect-symbiont interactions. However, endosymbiont research has focused on a few aphid species, making it necessary to make greater efforts to other aphid species through different regions, in order to have a better understanding of the role of endosymbionts in aphids as a group. Aphid endosymbionts have frequently been studied by PCR-based techniques, using species-specific primers, nevertheless this approach may omit other non-target bacteria cohabiting a particular host species. Advances in high-throughput sequencing technologies are complementing our knowledge of microbial communities by allowing us the study of whole microbiome of different organisms. We used a 16S rRNA amplicon sequencing approach to study the microbiome of aphids in order to describe the bacterial community diversity in introduced populations of the cereal aphids, Sitobion avenae and Rhopalosiphum padi in Chile (South America). An absence of secondary endosymbionts and two common secondary endosymbionts of aphids were found in the aphids R. padi and S. avenae, respectively. Of those endosymbionts, Regiella insecticola was the dominant secondary endosymbiont among the aphid samples. In addition, the presence of a previously unidentified bacterial species closely related to a phytopathogenic Pseudomonad species was detected. We discuss these results in relation to the bacterial endosymbiont diversity found in other regions of the native and introduced range of S. avenae and R. padi. A similar endosymbiont diversity has been reported for both aphid species in their native range. However, variation in the secondary endosymbiont infection could be observed among the introduced and native populations of the aphid S. avenae, indicating that aphid-endosymbiont associations can vary across the geographic range of an aphid species. In addition, we discuss the potential role of aphids as vectors and/or alternative hosts of phytopathogenic bacteria.

Project description:Forest soils provide a multitude of habitats for diverse communities of bacteria. In this study, we selected three tropical forests in Kenya to determine the diversity and community structure of soil bacteria inhabiting these regions. Kakamega and Irangi are rainforests, whereas Gazi Bay harbors mangrove forests. The three natural forests occupy different altitudinal zones and differ in their environmental characteristics. Soil samples were collected from a total of 12 sites and soil physicochemical parameters for each sampling site were analyzed. We used an amplicon-based Illumina high-throughput sequencing approach. Total community DNA was extracted from individual samples using the phenol-chloroform method. The 16S ribosomal RNA gene segment spanning the V4 region was amplified using the Illumina MiSeq platform. Diversity indices, rarefaction curves, hierarchical clustering, principal component analysis (PCA), and non-metric multidimensional scaling (NMDS) analyses were performed in R software. A total of 13,410 OTUs were observed at 97% sequence similarity. Bacterial communities were dominated by Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, and Acidobacteria in both rainforest and mangrove sampling sites. Alpha diversity indices and species richness were higher in Kakamega and Irangi rainforests compared to mangroves in Gazi Bay. The composition of bacterial communities within and between the three forests was also significantly differentiated (R = 0.559, p = 0.007). Clustering in both PCA and NMDS plots showed that each sampling site had a distinct bacterial community profile. The NMDS analysis also indicated that soil EC, sodium, sulfur, magnesium, boron, and manganese contributed significantly to the observed variation in the bacterial community structure. Overall, this study demonstrated the presence of diverse taxa and heterogeneous community structures of soil bacteria inhabiting three tropical forests of Kenya. Our results also indicated that variation in soil chemical parameters was the major driver of the observed bacterial diversity and community structure in these forests.

Dataset Information

The bias associated with amplicon sequencing does not affect the quantitative assessment of bacterial community dynamics.

Publications

The bias associated with amplicon sequencing does not affect the quantitative assessment of bacterial community dynamics.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets